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Journal of Lipid Research, Vol. 50, 1497-1504, July 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

Purification of recombinant apolipoproteins A-I and A-IV and efficient affinity tag cleavage by tobacco etch virus protease

Matthew R. Tubb², Loren E. Smith² and W. Sean Davidson¹

Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507
² M. R. Tubb and L. E. Smith contributed equally to this work.

This work was supported by the National Heart, Lung and Blood Institute of the National Institutes of Health Grants HL-62542, HL-67093, and HL-082734 (WSD), and a predoctoral fellowship from the Ohio Valley Affiliate of the American Heart Association (MRT).

Published, JLR Papers in Press, March 24, 2009.

¹ To whom correspondence should be addressed. email: sean.davidson{at}uc.edu

The expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. The ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. A common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. A specific protease can then cleave the tag and yield the native recombinant protein. However, the application of this strategy to apolipoproteins has proven somewhat problematic because of the tendency for these highly flexible proteins to be nonspecifically cleaved at undesired sites within the native protein. Although systems have been developed using a variety of proteases, many suffer from low yield and, especially, the high cost of the enzyme.We developed a method that utilizes the tobacco etch virus protease to cleave a histidine-tag from apolipoproteins A-I and A-IV expressed in Escherichia coli. This protease can be easily and inexpensively expressed within most laboratories. We found that the protease efficiently cleaved the affinity tags from both apolipoproteins without nonspecific cleavage. All structural and functional measurements showed that the proteins were equivalent to native or previously characterized protein preparations. In addition to cost-effectiveness, advantages of the tobacco etch virus protease include a short cleavage time, low reaction temperature, and easy removal using the protease's own histidine-tag.

Supplementary key words bacteria • expression • inexpensive • cost effective • low temperature cleavage • TEV

Abbreviations: ABCA1, ATP-binding cassette transporter A1; apo, apolipoprotein; CD, circular dichroism; CVD, cardiovascular disease; DMPC, dimyristoyl-phospatidylcholine; E. coli, Escherichia coli; Gly, glycine; HIC, hydrophobic interaction column; His-tag, histidine-tag; igase, IgA protease; IMAC, nickel affinity column; IPTG, isopropyl β-D-1-thiogalactopyranoside; LB, Luria broth; MLV, multilamellar vesicles; Pro, proline; STB, standard tris buffer; TEV, tobacco etch virus; Thr, threonine; Trp, tryptophan

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